Goat anti mouse igg h l hrp ab205719

The heart was homogenized in a buffer solution (T-PER Tissue Protein Extraction Reagent; Thermo Scientific, Rockford, USA). The extracted proteins were quantified (TaKaRa BCA Protein Assay Kit, Takara Holdings Inc, Japan), and the amount of protein to be applied to the gel was adjusted. The protein samples (1 μg of protein) were electrophoresed on 10% SDS-PAGE and transferred to the PVDF membrane. The membrane was blocked for 1 h (BLOCK ACE^®, MEGMILK SNOW BRAND, Japan) and incubated with primary antibodies (eNOS, 1:1,000, Purified Mouse Anti-eNOS/NOS Type III, BDbioscience, USA; GAPDH, 1:20,000, Anti-GAPDH Loading Control ab8245, Abcam, UK) at 4°C overnight. After washing with Phosphate Buffered Saline with Tween (PBST) buffer, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (1:5,000, Goat Anti-Mouse IgG H&L HRP ab205719, Abcam, UK) for 1 h at room temperature. Blots were washed with PBST, and immunoreactive bands were detected using an enhanced chemiluminescence system (ImmunoStar^®, FUJIFILM Wako Chemical Corporation, Japan) (Figure 1A). Optical density for individual bands was examined using the Fluor Chem FC2 (Cell Biosciences, Santa Clara, CA, United States of America). The densitometry ratios of eNOS to GAPDH were then computed.